Differentiation of bone marrow cells into neuronal cells and uses therefor

ABSTRACT

The present invention relates to methods of inducing differentiation of mammalian bone marrow stromal cells into neuronal cells by contacting marrow stromal cells with a neuronal differentiation-inducing compounds. Neuronal differentiation-inducing compounds of the invention include anti-oxidants such as, but not limited to, beta-mercaptoethanol, dimethylsulfoxide, butylated hydroxyanisole, butylated hydroxytoluene, ascorbic acid, dimethylfumarate, and n-acetylcysteine. Once induced to differentiate into neuronal cells, the cells can be used for cell therapy, gene therapy, or both, for treatment of diseases, disorders, or conditions of the central nervous system.

BACKGROUND OF THE INVENTION

Pluripotent stem cells have been detected in multiple tissues in theadult mammal, participating in normal replacement and repair, whileundergoing self-renewal (Hay, 1966, Regeneration, Holt, Rinehart andWinston, New York; McKay, 1999, Nature Med. 5:261-262; Lemiscka, 1999,Ann. N.Y. Acad. Sci. 872:274-288; Owens and Friedenstein, 1988, CibaFoundation Syp. 136, Chichester, U.K. pp. 42-60; Prockop, 1997, Science276:71-74; Ferrari et al., 1998, Science 279:1528-1530; Caplan, 1991, J.Orthop. Res. 9:641-650; Pereira et al., 1995, Proc. Natl. Acad. Sci. USA92:4857-4861; Kuznetsov et al., 1997, Brit. J. Haemotology 97:561-570;Majumdar et al., 1998, J. Cell Physiol. 176:57-66; Pittenger et al.,1999, Science 284:143-147). A subclass of bone marrow stem cells is oneprototype, capable of differentiating into osteogenic, chondrogenic,adipogenic and other mesenchymal lineages in vitro (Owens andFriedenstein, 1988, Ciba Foundation Symp. 136, Chichester, U.K. pp.42-60; Prockop, 1997, Science 276; 71-74; Ferrari et al., 1998, Science279:1528-1530; Caplan, 1991, J. Orthop. Res. 9:641-650; Pereira et al.,1995, Proc. Natl. Acad. Sci. USA 92:4857-4861; Kuznetsov et al., 1997,Brit. J. Haemotology 97:561-570; Majumdar et al., 1998, J. Cell.Physiol. 176:57-66; Pittenger et al., 1999, Science 284:143-147). Thesepluripotent cells have been termed marrow stromal cells (MSCs), andrecently have been used clinically to treat osteogenesis imperfecta(Horwitz et al., 1999, Nature Med. 5:309-313).

The recent discovery of stem cell populations in the central nervoussystem (CNS) has generated intense interest, since the brain has longbeen regarded as incapable of regeneration (Reynolds and Weiss, 1992,Science 255:1707-1710; Richards et al., 1992, Proc. Natl. Acad. Sci. USA89:8591-8595; Morshead et al., 1994, Neuron 13:1071-1082). Neural stemcells (NSCs) are capable of undergoing expansion and differentiatinginto neurons, astrocytes and oligodendrocytes in vitro (Reynolds andWeiss, 1992, Science 255:1707-1710; Johansson et al., 1999, Cell96:25-34; Gage et al., 1995, Annu. Rev. Neurosci. 18:159-192; Vescovi etal., 1993, Neuron 11:951-966). NSCs back transplanted into the adultrodent brain survive and differentiate into neurons and glia, raisingthe possibility of therapeutic potential (Lundberg et al., 1997, Exp.Neurol. 145:342-360; Lundberg et al., 1996, Brain Res. 737:295-300;Renfranz et al., 1991, Cell 66:713-729; Flax et al., 1998, NatureBiotech. 16:1033-1039; Gage et al., 1995, Proc. Natl. Acad. Sci. USA92:11879-11883; Svendsen et al., 1997, Exp. Neurol. 148:135-146).However, the inaccessibility of NSC sources deep in the brain severelylimits clinical utility. The recent report demonstrating that NSCs cangenerate hematopoietic cells in vivo suggests that stem cell populationsmay be less restricted than previously thought (Bjornson, 1999, Science283:534-537).

Evidence that MSCs injected into the lateral ventricles of neonatal micecan differentiate to astrocytes and neurofilament-containing cells lendssupport to this contention (Kopen et al., 1999, Proc. Natl. Acad. Sci.96:10711-10716).

However, although differentiation of MSCs into astrocytes and glialcells had been demonstrated (WO 99/43286), to date, there has been nomethod for inducing MSCs to differentiate into neuronal cells. Thus,despite the crucial need for obtaining neuronal cells for treatment ofCNS diseases, disorders, and conditions, no method has been availablefor obtaining large numbers of neuronal cells without encountering thetechnical and ethical hurdles involved in obtaining human NSCs or fetaltissue. The present invention overcomes this need.

BRIEF SUMMARY OF THE INVENTION

The present invention includes a method of inducing differentiation ofan isolated marrow stromal cell into a neuronal cell. The methodcomprises contacting the isolated marrow stromal cell with at least oneneuronal differentiation-inducing compound. This induces differentiationof the isolated marrow stromal cell into a neuronal cell.

In one aspect, the isolated marrow stromal cell is a rat cell.Preferably, the isolated marrow stromal cell is a human cell.

In one aspect, the neuronal differentiation-inducing compound is ananti-oxidant. In another aspect, the anti-oxidant is selected from thegroup consisting of beta-mercaptoethanol, dimethylsulfoxide, butylatedhydroxytoluene, butylated hydroxyanisole, ascorbic acid,dimethylfumarate, and n-acetylcysteine.

In yet another aspect, the anti-oxidant is beta-mercaptoethanol.

In another aspect, the anti-oxidant is dimethylsulfoxide. In yet anotheraspect the anti-oxidant is dimethylsulfoxide and butylatedhydroxyanisole.

The neuronal differentiation-inducing compound is also a growth factorin another aspect. In a preferred aspect, the growth factor is selectedfrom the group consisting of platelet-derived growth factor, fibroblastgrowth factor 2 and nerve growth factor.

The invention further includes a method of producing an isolatedneuronal cell. The method comprises isolating a marrow stromal cell,contacting the marrow stromal cell with a neuronaldifferentiation-inducing compound wherein the compound induces theisolated marrow stromal cell to differentiate into an isolated neuronalcell, thereby producing an isolated neuronal cell.

In addition, the invention includes a method of treating a human patienthaving a disease, disorder or condition of the central nervous system.The method comprises obtaining a bone marrow sample from a human donor,isolating stromal cells from the bone marrow sample, inducing thestromal cells to differentiate into isolated neuronal cells, andadministering the isolated neuronal cells to the central nervous systemof the human patient. The presence of the isolated neuronal cells in thecentral nervous system of the human patient effects treatment of thedisease, disorder or condition.

In one aspect, the disease, disorder or condition of the central nervoussystem is selected from the group consisting of Alzheimer's disease,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, a tumor, a trauma, elderly dementia, Tay-Sach's disease,Sandhoff's disease, Hurler's syndrome, Krabbe's disease, birth-inducedtraumatic central nervous system injury, epilepsy, multiple sclerosis,trauma, tumor, stroke, and spinal cord injury.

In another aspect, prior to administering the isolated neuronal cells,the isolated neuronal cells are transfected with an isolated nucleicacid encoding a therapeutic protein, wherein when the protein isexpressed in the cells the protein serves to effect treatment of thedisease, disorder or condition.

In an alternative aspect, the isolated neuronal cells are transfectedwith an isolated nucleic acid encoding a cytokine, a chemokine, aneurotrophin, another trophic protein, a growth factor, an antibody, orglioma toxic protein.

The present invention further includes a method of treating a humanpatient in need of neuronal cells. The method comprises obtaining marrowstromal cells from a human patient, propagating the marrow stromal cellsin culture under conditions that induce their differentiation intoneuronal cells, transplanting the neuronal cells into the human patientin need of the neuronal cells, thereby treating the human patient inneed of neuronal cells.

The invention also includes an isolated neuronal cell made by the methodof inducing differentiation of an isolated marrow cell into a neuronalcell. The method comprises contacting the isolated marrow stromal cellwith at least one neuronal differentiation-inducing compound. Thecontact between the isolated marrow stromal cell and the neuronaldifferentiation-inducing compound induces differentiation of theisolated marrow stromal cell into the neuronal cell of the invention.

In an aspect, the neuronal cell made by this method is a rodent cell. Inanother aspect, the neuronal cell is a rat cell. In a preferred aspect,the neuronal cell made by this method is a human neuronal cell.

A preferred embodiment of the invention includes an isolated neuronalcell transfected with a therapeutic protein. The neuronal cell isisolated by the method of inducing differentiation of an isolated marrowcell into a neuronal cell recited above. The neuronal cell is thentransfected with an isolated nucleic acid encoding a therapeutic proteinthat when expressed, will effect treatment of a disease, disorder, orcondition of the central nervous system. In an aspect of the invention,the therapeutic protein encoded by the isolated nucleic acid is acytokine, a chemokine, a neurotrophin, another trophic protein, a growthfactor, an antibody, or glioma toxic protein.

The invention encompasses diseases, disorders, or conditions of thecentral nervous system including, but are not limited to, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, a tumor, a trauma, elderly dementia, Tay-Sach's disease,Sandhoff's disease, Hurler's syndrome, Krabbe's disease, birth-inducedtraumatic central nervous system injury, epilepsy, multiple sclerosis,trauma, tumor, stroke, and spinal cord injury.

In one aspect, the transfected neuronal cell made by the method ofinducing differentiation of an isolated marrow stromal cell is a ratcell or a rodent cell. Preferably the transfected neuronal cell is ahuman cell.

The invention also includes an isolated neuronal cell produced by amethod comprising isolating a marrow stromal cell and contacting it witha neuronal differentiation-inducing compound. This induces the isolatedmarrow stromal cell to differentiate into isolated neuronal cells.

A transfected isolated neuronal cell produced by isolating a marrowstromal cell and contacting it with a neuronal differentiation-inducingcompound is also included in the invention. The isolated neuronal cellproduced by this method is then transfected with an isolated nucleicacid encoding a therapeutic protein, that, when expressed in theneuronal cell, will effect treatment of a disease, disorder, orcondition of the central nervous system.

In a preferred aspect, the therapeutic protein encoded by the isolatednucleic acid is a cytokine, a chemokine, a neurotrophin, another trophicprotein, a growth factor, an antibody, or a glioma-toxic protein.

In an aspect of the present invention, the transfected neuronal cellproduced by contacting a neuronal differentiation-inducing compound witha marrow stromal cell is a rat cell. In another aspect, the transfectedneuronal cell is a rodent cell. In a preferred aspect, the transfectedneuronal cell is a human cell.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiment(s) which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown. In thedrawings:

FIG. 1A is a graph depicting fluorescent cell sorting of passage 1 rMSCsusing mouse monoclonal antibodies that specifically bind with cellsurface marker CD11b (CD11/integrin alpha_(M)/Mac-1 alpha chain;Pharmingen, San Diego, Calif.) (unfilled peaks). The secondary antibodyused was anti-mouse antibody conjugated with fluoresceine isothiocyanate(FITC). An isotype control is included in each experiment to identifybackground fluorescence (filled peaks). Number of cells analyzed(Events) is plotted on the Y-axis, while intensity of staining isplotted on the X-axis.

FIG. 1B is a graph depicting fluorescent cell sorting of passage 1 rMSCsusing mouse monoclonal antibodies that specifically bind with cellsurface marker CD45/leukocyte common antigen (Pharmingen) (unfilledpeaks). The secondary antibody is anti-mouse antibody conjugated withfluoresceine isothiocyanate (FITC). An isotype control is included ineach experiment to identify background fluorescence (filled peaks).Number of cells analyzed (Events) is plotted on the Y-axis, whileintensity of staining is plotted on the X-axis.

FIG. 1C is a graph depicting fluorescent cell sorting of passage 1 rMSCsusing mouse monoclonal antibodies that specifically bind with cellsurface marker CD90/Thy-1/CD90.1/Thy1.1 (Pharmingen) (unfilled peaks).The secondary antibody is anti-mouse antibody conjugated withfluoresceine isothiocyanate (FITC). An isotype control is included ineach experiment to identify background fluorescence (filled peaks).Number of cells analyzed (Events) is plotted on the Y-axis, whileintensity of staining is plotted on the X-axis. The data disclosedherein demonstrate that the fluorescence intensity is greater (shiftedto the right) when rMSCs are incubated with CD90 antibody (unfilled), ascompared to control antibody (filled), indicating that the vast majorityof cells in the rMSC cultures express CD90, consistent with theirundifferentiated state.

FIG. 2, comprising FIGS. 2A-2H, is an image depicting neuronaldifferentiation of rMSCs at various time points after treatment.Briefly, the neuronal differentiation protocol disclosed herein wasinitiated at 0 minutes and followed for 210 minutes. FIG. 2A represents0 minutes, FIG. 2B represents 30 minutes, FIG. 2C represents 60 minutes,FIG. 3D represents 90 minutes, FIG. 2E represents 120 minutes, FIG. 2Frepresents 150 minutes, FIG. 2G represents 180 minutes, and FIG. 2Hrepresents 210 minutes. A flat rMSC in FIG. 2A is identified (▾) priorto differentiation. Retraction of cell body and process elaboration isevident with increasing time. The arrow in FIG. 2E indicates a seconddifferentiating cell. Retracting neurite is indicated by (>).(Magnification=200×).

FIG. 3A is an image depicting neuron-specific enolase (NSE) expressionin differentiating neurons using an anti-NSE polyclonal antibody(Polysciences, Warrington, Pa.). Briefly, undifferentiated rMSCs(indicated by a “>”) retained flattened morphology and stained onlyslightly for NSE expression. rMSC-derived neurons (arrows) stained darkbrown for NSE expression and displayed condensed cell bodies and highlybranches processes. Transitional cells (

) exhibited intermediate neuronal morphologies, with partially retractedcell bodies and light brown NSE staining.

FIG. 3B is an image depicting that the morphologies of rMSC-derivedneurons include simple bipolar (▾) and complex multipolar cells withhighly branched processes (arrow). Intense NSE staining is evident inboth neuronal cell types.

FIG. 3C is an image depicting that NSE-positive neurons displayingpyramidal morphologies are sometimes generated using the protocolsdisclosed elsewhere herein. Contact with a transitional cell (lightbrown) is maintained via a single unbranched process.

FIG. 3D is an image depicting a NSE-positive neuron elaborating a longprocess with evident varicosities (arrows). The data disclosed hereindemonstrate that the neuronal cell body is in intimate contact with atransitional cell.

FIG. 3E is an image depicting that clusters of rMSC-derived neurons ofvarying morphologies form complex networks. The data disclosed hereindemonstrate that an undifferentiated rMSC (>) is included within thismeshwork of processes. (Magnification=320×).

FIG. 3F is an image of a Western blot analysis disclosing expression oflow levels of NSE in uninduced rMSCs (U). The data disclosed hereindemonstrate that a significant increase in NSE expression is evident at5 hours post BME treatment (I). Comparable levels of tubulin aredetected in each lane, indicating equal loading of samples.

FIG. 4A is an image depicting NeuN expression in rMSC-derived neuronsusing a monoclonal anti-NeuN antibody (Chemicon, Temeeula, Calif.).Briefly, the data disclosed herein demonstrate that NeuN can be detectedin the nucleus and surrounding cytoplasm of rMSC-derived neurons(arrow).

FIG. 4B is an image depicting NeuN expression in rMSC-derived neuronsusing a monoclonal anti-NeuN antibody (Chemicon). Briefly, the datadisclosed herein demonstrate that NeuN can be detected in the nucleusand surrounding cytoplasm of rMSC-derived neurons (arrow). Further, thedata disclosed herein demonstrate that anti-NeuN antibody staining doesnot extend into the processes of positive cells. The image furtherdepicts that transitional cells (

) and undifferentiated rMSCs (<) do not express NeuN.

FIG. 5A is an image depicting expression of NF-M and tau bydifferentiating cells. Briefly, rMSC-derived neurons were immunostainedto detect expression of NF-M using an anti-NF-M polyclonal antibody(Chemicon). The data disclosed herein demonstrate that cells thatexhibit neuronal morphologies express NF-M in both cell bodies (arrow)and processes (*). Flat, undifferentiated rMSCs (>) do not stain forNF-M expression.

FIG. 5B is an image depicting that pre-adsorption of anti-NF-M antibody(Chemicon) with 20 micrograms of purified NF-M protein overnight at 4°C. eliminated staining of rMSC-derived neurons, indicating specificityof the NF-M staining.

FIG. 5C is an image depicting rMSC-derived neurons stained forexpression of tau using anti-tau polyclonal antibody (Sigma ChemicalCo., St. Louis, Mo.). The data disclosed herein demonstrate that cellsdisplaying neuronal morphologies (arrows) stain dark brown for tauexpression within the cell body and extending into the processes (*).Flat, undifferentiated rMSCs (>) do not express tau and are unstained.(Magnification=320×).

FIG. 5D is an image depicting rMSC-derived neurons stained forexpression of tau using anti-tau polyclonal antibody (Sigma ChemicalCo., St. Louis, Mo.). The data disclosed herein demonstrate that cellsdisplaying neuronal morphologies (arrows) stain dark brown for tauexpression within the cell body and extending into the processes (*).Flat, undifferentiated rMSCs (>) do not express tau and are unstained.(Magnification=320×).

FIG. 6A is an image depicting FM1-43 labeling of rMSC-derived neurons.The data disclosed herein demonstrate that rMSC-derived neuronsdepolarized using KCl demonstrate intense labeling of terminal putativegrowth cones (indicated by unfilled triangle).

FIG. 6B is an image depicting FM1-43 labeling of rMSC-derived neurons.The data disclosed herein demonstrate that rMSC-derived neuronsdepolarized using KCl demonstrate intense labeling of terminal putativegrowth cones (indicated by unfilled triangle).

FIG. 7A is an image depicting differentiation of clonal rMSC lines.NSE-staining of individual rMSC clone #1 subjected to thedifferentiation protocol disclosed herein. NSE-positive cells (darkbrown) are derived from each clonal line. Undifferentiated rMSCs (>)and/or transitional cells (

) are evident in each panel. (Magnification=320×).

FIG. 7B is an image depicting differentiation of clonal rMSC lines.NSE-staining of individual rMSC clone #2 subjected to thedifferentiation protocol disclosed herein. NSE-positive cells (darkbrown) are derived from each clonal line. Undifferentiated rMSCs (>)and/or transitional cells (

) are evident in each panel. (Magnification=320×).

FIG. 7C is an image depicting differentiation of clonal rMSC lines.NSE-staining of individual rMSC clone #3 subjected to thedifferentiation protocol disclosed herein. NSE-positive cells (darkbrown) are derived from each clonal line. Undifferentiated rMSCs (>)and/or transitional cells (

) are evident in each panel. (Magnification=320×).

FIG. 7D is an image depicting differentiation of clonal rMSC lines.NSE-staining of individual rMSC clone #1 subjected to thedifferentiation protocol disclosed herein. NSE-positive cells (darkbrown) are derived from each clonal line. Undifferentiated rMSCs (>)and/or transitional cells (

) are evident in each panel. (Magnification 320×).

FIG. 8A is an image depicting differentiation of human MSCs. The datadisclosed herein demonstrate that human MSCs differentiate into neuronsand express high levels of NSE (dark brown). A lighter stainedtransitional cell (indicated by

) is depicted at lower left.

FIG. 8B is an image depicting that an NSE-positive hMSC-derived neuronelaborates a process exhibiting neuronal-like terminal bulb morphology.

FIG. 8C is an image depicting a phase-contrast image of pairedNSE-positive neurons. The data disclosed herein demonstrate growth conemorphologies with filopodial extensions (double arrow). The image isenlarged 50% to show detail.

FIG. 8D is an image depicting that hMSC-derived neurons stain positivefor NF-M. (Magnification=320×).

FIG. 9, comprising FIGS. 9A-9F, is an image depicting nestin and trkAexpression in differentiating rMSC-derived neurons. FIGS. 9A-9Cdemonstrate cells stained for nestin expression at 5 hours, 1 day, and 6days, respectively. FIGS. 9D-9F represent cells stained for trkAexpression at 5 hours, 1 day, and 6 days, respectively.(Magnification=320×).

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery that contacting marrow stromalcells with a neural-differentiation inducing agent mediatesdifferentiation of the cells into neuronal-like cells expressing avariety of neuron-specific markers (e.g., NeuN, neurofilament-M,neuron-specific enolase [NSE], tau, nestin, trkA, and the like). Thecells exhibit other neuron-like phenotypic characteristics such as, butnot limited to, spherical and refactile cell bodies exhibiting typicalneuronal perikaryal appearance, cell bodies extending long processesterminating in growth cones and filopodia typical of neurons, andlabeling of the growth cones by the fluorescent dye FM1-43, whichtypically labels neuronal transmitter release and synaptic vesiclerecycling. Thus, the methods disclosed herein induce marrow stromal celldifferentiation into neuronal cells. Such methods are crucial in thedevelopment of cell-based therapeutics for treatment of central nervoussystem (CNS) disorders, diseases or conditions. Indeed, prior to thepresent invention, the lack of source of neuronal cells, which can beintroduced into the CNS of a human patient, has severely impeded thedevelopment of CNS therapeutics.

DESCRIPTION

The invention includes a method of inducing an isolated marrow stromalcell to differentiate into an isolated neuronal cell. Embodiments of themethod of the invention are described in the Examples section herein.Generally, cells are isolated from a donor, stromal cells are obtainedtherefrom, usually using a cell-sorting method, and the stromal cellsare subsequently cultured in vitro. The donor may be a rat, for example,or the donor may be a human. The invention is intended to encompass amammalian donor and should not be limited to the specific donorsdisclosed herein.

To induce the neuronal phenotype, the cells are pre-treated with aneffective amount of a neuronal differentiation-inducing compound whichis introduced into the cell culture for a period of time. The length oftime may vary according to the precise method being contemplated andshould not be construed as limiting the invention in any way. Afterpre-treatment exposure to the neuronal differentiation inducingcompound, the cells are transferred to a serum-free medium containing anamount of the same neuronal differentiation-inducing compound. Neuronalmorphology is evident within about an hour, see FIG. 2 for example, andthe morphology becomes more evident steadily over time. Neuronal markerexpression is also apparent within about 30 minutes after treatment.Neuronal cells so differentiated also eventually express several proteinmarkers, including but not limited to, tyrosine hydroxylase, tubulin,choline acetyltransferase, synaptophysin, and TOAD, which are allproteins necessarily associated with neurons and neuronal processes.

These newly differentiated neuronal cells are useful in treatingpatients afflicted with diseases of the cholinergic andcatecholaminergic systems, and more generally, patients afflicted withdiseases of the central nervous system.

In one embodiment of the invention, antioxidants serve as the neuronaldifferentiation-inducing compounds, including but not limited to,beta-mercaptoethanol, dimethylsulfoxide, butylated hydroxytoluene,butylated hydroxyanisole, ascorbic acid, dimethylfumarate, andn-acetylcysteine. Particularly preferred embodiments as demonstrated inthe Examples section herein disclosed, include beta-mercaptoethanol,dimethylsufloxide and a combination of dimethylsulfoxide and butylatedhydroxyanisole as the favored antioxidants. However, the invention isnot limited to those antioxidants disclosed herein and should beconstrued to include all antioxidants, as well as other compounds whichinduce neuronal differentiation of marrow stromal cells.

The invention also contemplates use of growth factors as the neuronaldifferentiation-inducing compounds in the method of inducingdifferentiation of MSCs to neuronal cells. Such growth factors include,but are not limited to fibroblast growth factor 2, platelet-derivedgrowth factor, and nerve growth factor, as well as related agents.

Neuronal identity can be confirmed by staining the differentiatedneuronal cells for detection of neuron-specific markers. Examples ofsuch markers are neurofilament-M (NF-M), tau protein, Neu-N,neuron-specific enolase USE), nestin, and trkA. Progressivedifferentiation of the marrow stromal cell to the neuronal cellcorresponds with an increase in each of these markers, indicating thatneuronal cells are produced. Further characterization can beaccomplished using known immunocytochemical and antibody techniques. Forexample, immunocytochemical analysis of these neuronal cells revealsthat the cells also express proteins that are associated withnaturally-differentiated neurons. Such proteins include, but are notlimited to tubulin, TOAD, and synaptophysin. Antibody detection ofcholine acetyltransferase and tyrosine hydroxylase may also be assessed.

It is apparent from the data disclosed herein that it is possible todifferentiate isolated marrow stromal cell into neuronal cells in vitro.Neuronal cells so differentiated are useful in treating patientsafflicted with any of a wide variety of central nervous system diseases,disorders, or conditions.

The invention also includes a method for producing an isolated neuronalcell from isolated marrow stromal cells. The method comprisesdifferentiating an isolated marrow stromal cell in the same generalmanner as recited above, thereby producing an isolated neuronal cell.

The isolated neuronal cell recited in both of the methods above may betransfected with an isolated nucleic acid encoding a therapeuticprotein. The therapeutic protein, when expressed, will treat a patienthaving a disease, disorder, or condition of the central nervous system.

A wide plethora of beneficial proteins are well-known in the art and areset forth in, for example, WO 96/30031 and WO 99/43286. Such examplesinclude, but are not limited to, cytokines, chemokines, neurotrophins,other trophic proteins, growth factors, antibodies, and glioma toxicprotein. When the transfected neuronal cells encoding such proteins areadministered to a patient, the neuronal cells will beneficiallyinfluence cells which are already present in the central nervous system.For example, transfected neuronal cells which are introduced into thecentral nervous system may be used to repair any central nervous systemdamage, and/or to combat tumors of the central nervous system.

International patent applications WO 96/30031 and WO 99/43286 alsodescribe use of MSCs in therapies for a wide variety of CNS diseases,disorders, or conditions, which include, but are not limited to, geneticdiseases of the CNS (e.g., Tay-Sach's, Sandhoff's disease, Hurler'ssyndrome, Krabbe's disease), birth-induced traumatic CNS injury, adultCNS diseases, disorders or conditions (e.g., Parkinson's, Alzheimer's,and Huntington's diseases, elderly dementia, epilepsy, amyotropiclateral sclerosis, multiple sclerosis, trauma, tumors, stroke, and thelike) and degenerative diseases and traumatic injury of the spinal cord.

Among neonates and children, transfected neuronal cells may be used fortreatment of a number of genetic diseases of the central nervous system,including, but not limited to, Tay-Sachs disease and the relatedSandhoff's disease, Hurler's syndrome and related mucopolysaccharidosesand Krabbe's disease. To varying extents, these diseases also producelesions in the spinal cord and peripheral nerves and they also havenon-neurological effects. While the non-neurological effects of thesediseases may be treatable by bone marrow transplantation, the centralnervous system effects do not improve despite bone marrowtransplantation. The method of the present invention is useful toaddress the central nervous system effects of these types of diseases.In addition, in neonates and children, head trauma during birth orfollowing birth is treatable by introducing these neuronal cellsdirectly into the central nervous system of the children. Centralnervous system tumor formation in children is also treatable using themethods of the present invention.

Adult diseases of the central nervous system are also treatable byadministering isolated neuronal cells to the adult. Such adult diseasesinclude but are not limited to, Parkinson's disease, Alzheimer'sdisease, spinal cord injury, stroke, trauma, tumors, degenerativediseases of the spinal cord such as amyotropic lateral sclerosis,Huntington's disease and epilepsy. Treatment of multiple sclerosis isalso contemplated.

Treatment of spinal cord injuries is also possible using the method ofthe present invention Prior art methods of treating spinal cord injuriesinvolve using fibroblast cells to deliver neurotrophins to the site ofspinal cord lesions in animals. The neurotrophins, delivered in thismanner, reduce the lesion or otherwise treat the injury. However,fibroblasts produce large amounts of collagen, causing fibrosis at thesite of the lesion, thus negating the beneficial effects of thetreatment. Delivery of neurotrophins to spinal cord lesions usingtransfected neuronal cells is advantageous over prior art methodsbecause neuronal cells do not produce large amounts of collagen andtherefore should not cause fibrosis.

The invention further includes a method of treating a human patienthaving a disease, disorder, or condition of the central nervous systemby administering the differentiated neuronal cells of the invention tothe central nervous system of the patient. Methods of treating a humanpatient using MSCs are described in WO 96/30031 and WO 99/43286, whichare incorporated by reference as if set forth in their entirety herein.Methods of administering differentiated neuronal cells to a patient areidentical to those used for MSCs as described in WO 96/30031 and WO99/43286. The methods encompass introduction of an isolated nucleic acidencoding a beneficial protein into differentiated neuronal cells andalso encompass using differentiated neuronal cells themselves incell-based therapeutics where a patient is in need of the administrationof such cells. The differentiated neuronal cells are preferablyadministered to a human, and further, the neuronal cells are preferablyadministered to the central nervous system of the human. In someinstances, the differentiated neuronal cells are administered to thecorpus striatum portion of the human brain. The precise site ofadministration of the neuronal cells will depend on any number offactors, including but not limited to, the site of the lesion to betreated, the type of disease being treated, the age of the human and theseverity of the disease, and the like. Determination of the site ofadministration is well within the skill of the artisan versed in theadministration of cells to mammals.

The mode of administration of the differentiated neural cells to thecentral nervous system of the human may vary depending on severalfactors including but not limited to, the type of disease being treated,the age of the human, whether the neuronal cells have isolated DNAintroduced therein, and the like. An example of administration ofneuronal cells directly into brain tissue is provided herein in theexperimental details section. Generally, cells are introduced into thebrain of a mammal by first creating a hole in the cranium through whichthe cells are passed into the brain tissue. Cells may be introduced bydirect injection, by using a shunt, or by any other means used in theart for the introduction of compounds into the central nervous system.

DEFINITIONS

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, “central nervous system” should be construed to includebrain and/or the spinal cord of a mammal. The term may also include theeye and optic nerve in some instances.

As used herein, “stromal cells”, “isolated marrow stromal cells”, and“MSCs” are used interchangeably and are meant to refer to the smallfraction of cells in bone marrow which can serve as stem cell-likeprecursors of osteocytes, chondrocytes, and adipocytes and which areisolated from bone marrow by their ability adhere to plastic dishes.Marrow stromal cells may be derived from any animal. In someembodiments, stromal cells are derived from primates, preferably humans.

As used herein, the term “anti-oxidant” is meant to refer to thosesubstances that inhibit oxidation or reactions promoted by oxygen orperoxides. Examples of anti-oxidants include, but are not limited to,beta-mercaptoethanol, dimethylsulfoxide, butylated hydroxytoluene,butylated hydroxyanisole, ascorbic acid, dimethylfumarate, andn-acetylcysteine.

As used herein, the terms “beneficial protein” and “therapeutic protein”are used interchangeably and are meant to refer to a protein which cancompensate for the protein encoded by a defective gene and/orinsufficient gene expression that is causally linked to the disease orsymptoms of the disease, disorder or condition characterized by a genedefect. The presence of the protein alleviates, reduces, prevents orcauses to be alleviated, reduced or prevented, the causes and/orsymptoms that characterize the disease, disorder or condition.

As used herein, a disease, disorder or condition which can be treatedwith a beneficial or therapeutic protein is meant to refer to a disease,disorder or condition that can be treated or prevented by the presenceof a protein which alleviates, reduces, prevents or causes to bealleviated, reduced or prevented, the causes and/or symptoms thatcharacterize the disease, disorder or condition. Diseases, disorders andconditions which can be treated with a beneficial protein includediseases, disorders and conditions characterized by a gene defect aswell as those which are not characterized by a gene defect but whichnonetheless can be treated or prevented by the presence of a proteinwhich alleviates, reduces, prevents or causes to be alleviated, reducedor prevented, the causes and/or symptoms that characterize the disease,disorder or condition.

The term “isolated nucleic acid” should be construed to refer to anucleic acid sequence, or segment, or fragment which has been purifiedfrom the sequences which flank it in a naturally occurring state, e.g.,a DNA fragment which has been removed from the sequences which arenormally adjacent to the fragment e.g., the sequences adjacent to thefragment in a genome in which it naturally occurs. The term also appliesto nucleic acids which have been substantially purified from othercomponents which naturally accompany the nucleic acid, e.g., RNA or DNAor proteins which naturally accompany it in the cell.

As used herein, “transfected cells” is meant to refer to cells to whicha gene construct has been provided using any technology used tointroduce nucleic acid molecules into cells such as, but not limited to,classical transfection (calcium phosphate or DEAE dextran mediatedtransfection), electroporation, microinjection, liposome-mediatedtransfer, chemical-mediated transfer, ligand mediated transfer orrecombinant viral vector transfer.

The term “differentiation” as used herein, should be construed to meanthe induction of a differentiated phenotype in an undifferentiated cellby coculturing the undifferentiated cell in the presence of asubstantially homogeneous population of differentiated cells, in thepresence of products of differentiated cells or in the presence of aninducer of cell differentiation.

The term “neuronal cell” as used herein should be construed to mean anMSC differentiated such that it expresses at least one of the followingneuronal markers: neuron-specific enolase (NSE), NeuN, neurofilament M,or tau protein.

The term “neuron” as used herein should be construed to mean a nervecell capable of receiving and conducting electrical impulses from thecentral nervous system. A nerve cell or “neuron” typically comprises acell body, an axon, axon terminals, and dendrites.

The term “neuronal differentiation-inducing compound” is meant to referto those compounds capable of inducing differentiation of a stromal cellinto a neuronal cell. These compounds include, but are not limited toantioxidants, trophic factors, and growth factors.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

EXAMPLES

The experiments presented in this example may be summarized as follows.

Bone marrow stromal cells exhibit multiple traits of a stem cellpopulation. They can be greatly expanded in vitro, and induced todifferentiate into multiple mesenchymal cell types (see, e.g., WO96/30031; WO 99/43286). However, differentiation to non-mesenchymalfates has not been demonstrated. Here, adult rat stromal cells wereexpanded as undifferentiated cells in culture for more than 14 passages,indicating their proliferative capacity. Further, a novel treatmentprotocol induced the stromal cells to exhibit a neuronal phenotype,expressing various neuron-specific markers, i.e., neuron-specificenolase (NSE), NeuN, neurofilament-M, tau, nestin, and trkA.

Moreover, the retractile cell bodies of the treated cells extended longprocesses terminating in growth cones and filopodia typical of neuronalcells. The fluorescent dye, FM1-43, labeled growth cones, consistentwith transmitter release and synaptic vesicle recycling by the treatedcells. Clonal cell lines, established from single cells, proliferated,yielding both undifferentiated and cells exhibiting a neuronalphenotype.

Human marrow stromal cells treated using the novel protocol disclosedherein differentiated into neurons similarly to rMSCs demonstrating thatthe protocol is not limited to rodent stromal cells. Consequently, thedata disclosed herein demonstrate, for the first time, that mammalianmarrow stromal cells can be induced to overcome their mesenchymalcommitment, and can constitute an abundant and accessible cellularreservoir for the treatment of a variety of neurologic diseases,disorders or conditions.

The Materials and Methods used in the experiments presented in thisexample are now described.

Cell Culture

Rat MSCs were originally cultured in alpha-Modified Eagle's Medium(alpha-MEM) supplemented with 20% FBS, 2 mM L-glutamine, 100 units permilliliter penicillin, 100 milligrams per milliliter streptomycin and 25nanograms per milliliter amphotericin B. For each passage the cells wereplated at about 8,000 cells per square centimeter and grown toconfluency. At passage 6 the cells were transferred to DMEM (pH 8.0)/20%FBS without additional supplementation, and maintained beyond passage14. The rat MSCs were obtained with a protocol and procedures approvedby the Institutional IACUC. The human samples were obtained fromvolunteers with informed consent and according to a protocol approved bythe Institutional Review Board.

Western Blot

Thirty milligrams of protein extract from untreated (U) and BME-induced(I) rMSC cultures was separated on a 4%-20% gradient acrylamide gel andelectrophoretically transferred to a nylon membrane. The Western blotwas probed for tubulin expression using an anti-tubulin monoclonalantibody (Sigma Chemical. Co., St. Louis, Mo.) followed by secondaryantibody conjugated with horse radish peroxidase (HRP). Colordevelopment was performed using enhanced chemiluminescence reagents(Amersham, Piscataway, N.J.). The blot was then stripped and probed forNSE expression using anti-NSE polyclonal antibody (ICN). Again, thesecondary antibodies were HRP-conjugated, and color was developed usingECL reagents.

Immunocytochemistry

Cultured rMSCs were fixed with 4% paraformaldehyde, incubated withprimary antibody overnight at 4° C., incubated with secondary antibodyfor one hour, followed by exposure to avidin-biotin complex for one hourat 25° C. Diaminobenzidene (DAB) served as chromogenic substrate forHRP.

FM1-43 Labeling

Cultures were treated with DMSO/BI-IA in serum-free media (SFM) forapproximately 4 hours. The cells were maintained for an additional 30minutes in artificial cerebral spinal fluid (aCSF)/BHA. Cells werelabeled in aCSF containing 1 millimolar FM1-43 and 75 mM KCl for 60seconds. The labeling mixture was removed, the cultures were washedtwice with aCSF, and the cells were incubated in aCSF for 60 minutes toreduce background staining. Cultures were fixed with 4%paraformaldehyde, and soaked for 24 hours in phosphate buffered saline(PBS) before analysis.

The Results of the experiments presented in this example are nowdescribed.

Stromal Cell Characterization

Rat mesenchymal stromal cells (rMSCs) were isolated from the femurs ofadult rats and propagated in vitro (Azizi et al., 1998, Proc. Natl.Acad. Sci. USA 95:3908-3913). The data disclosed in FIG. 1A demonstratethat the distribution of cells stained with antibody to CD11b (unfilled)does not differ from that of isotype control (filled), indicating therMSC cultures do not contain significant numbers of contaminatingCD11b-expressing cells. Further, the data disclosed in FIG. 1B alsodemonstrate that the intensity of staining does not differ between CD45antibody (unfilled) and control (filled) profiles, indicating thatcultured rMSCs are not contaminated by CD45-expressing cells.Fluorescent cell sorting at passage one also demonstrated that the cellswere negative for CD11b (FIG. 1A), and CD45 (FIG. 1B), which are cellsurface markers associated with lymphohematopoietic cells. Therefore,there was no evidence of hematopoietic precursors in the cultures.

In contrast, the data disclosed herein demonstrate that rMSCs expressedCD90 (FIG. 1C), consistent with their undifferentiated state. At lowplating densities rMSCs grew as a monolayer of large, flat cells. As thecells approached confluency, they assumed a more spindle-shaped,fibroblastic morphology. At the outset of the neuronal differentiationstudies disclosed elsewhere herein, untreated rMSCs were furthercharacterized by staining for the cell surface markers CD44 and CD71.Cells were positive for CD44 and CD71 expression, consistent withprevious reports (Pittenger et al., 1999, Science 284:143-147; Bruder etal., 1998, Clin. Orthop. Relat. Res. 355S:S247-S256).

Neuronal Differentiation

To induce the neuronal phenotype, rMSCs were initially maintained insub-confluent cultures in media supplemented with 1 mMbeta-mercaptoethanol (BME) for 24 hours. Under these conditions nochanges in morphology were evident. To effect neuronal differentiation,the cells were transferred to serum-free medium containing 1-10millimolar BME (SFM/BME). The percentage of cells adopting a neuronalmorphology increased at higher BME concentrations, and was enhanced byBME pretreatment. Within 60 minutes of exposure to SFM/BME changes inmorphology of some of the rMSCs were apparent (FIG. 2C). Responsivecells progressively assumed neuronal morphological characteristics overthe first 3 hours. Initially, cytoplasm in the flat rMSCs retractedtowards the nucleus, forming a contracted multipolar, cell body, leavingmembranous, process-like extensions peripherally (0-90 minutes).

Treated cells exhibited increased expression of the neuronal marker NSEwithin 30 minutes of treatment. Over the subsequent 2 hours cell bodiesbecame increasingly spherical and refractile, exhibiting a typicalneuronal perikaryal appearance. Processes continued to elaborate,developing growth cone-like terminal expansions and filopodialextensions (see, e.g., FIGS. 2G and 2H). Cellular processes exhibitedprimary and secondary branches, and underwent dynamic growth.Retraction, as well as extension, was evident as demonstrated by thefact that the cell marked by an arrow at 120 minutes (FIG. 2E) wasinitially contacted by a neighboring process (marked by “>”), whichretracted by 180 minutes (FIG. 2G), with loss of contact.

To further characterize potential neuronal differentiation, BME-treatedcultures were stained to detect expression of the neuronal markerneuron-specific enolase (NSE). Unresponsive, flat rMSCs expressed verylow, but detectable, levels of NSE protein, consistent with previousdetection of minute amounts of protein and/or message in cells of bonemarrow origin (Pechumer et al., 1993, Lab. Invest. 89:743-749; Reid etal., 1991, Clin. Pathol. 44:483-486; vanObberghen et al., 1988, J.Neurosci. Res. 19:450-456).

Progressive transition of rMSCs to a neuronal phenotype coincided withincreased expression of NSE (FIG. 3A). Cells that exhibited contractedcell bodies and processes stained dark brown for NSE expression(arrows), while flat, unresponsive rMSCs (>) displayed minimal NSEstaining. Cells at intermediate stages in the differentiation sequence (

) exhibited transitional morphologies and light brown staining,indicating synchrony of morphologic and molecular differentiation.rMSC-derived neurons displayed distinct neuronal morphologies (FIG. 3B),ranging from simple bipolar (▾) to large, extensively branchedmultipolar cells (arrow). Rare NSE-positive neurons exhibited pyramidalcell morphologies (FIG. 3C), while neurons elaborating long processeswith evident varicosities (arrows) were more common (FIG. 3D). Clustersof differentiated cells exhibited intense NSE positivity, and processesformed extensive networks (FIG. 3E). Even within these clusters,typical, flat rMSCs (>) were only lightly stained, consistent with theirundifferentiated state.

Western blot analysis (FIG. 3F) confirmed the expression of low levelsof NSE protein in uninduced rMSCs. Induction of the neuronal phenotyperesulted in a dramatic increase in NSE expression, consistent with theimmunocytochemical data.

To further investigate neuronal characteristics, differentiated cultureswere stained for NeuN, a neuron-specific marker expressed inpost-mitotic cells (Sarnat et al., 1998, Brain Res. 20:88-94). A subsetof cells exhibiting rounded cell-bodies and processes (arrow) stainedfor NeuN expression, while undifferentiated cells (<) remainedNeuN-negative (FIG. 4A). Consistent with previous reports describingNeuN staining of neuronal cells (Sarnat et al., 1998, Brain Res.20:88-94), NeuN staining was confined to the nucleus and surroundingcytoplasm of positive cells, and did not extend into the processes. Somecells exhibiting distinct neuronal morphologies did not express NeuN (

), while neighboring cells were intensely positive (arrow) (FIG. 4B).This pattern contrasts with that established for NSE staining, whereevery cell exhibiting a neuronal morphology demonstrated increased NSEexpression. Without wishing to be bound by any particular theory, thesedata suggest that a subset of NSE-positive cells are post-mitoticneurons. Also without wishing to be bound by any particular theory, itmay be that the anti-oxidant properties of BME, which enhance neuronalsurvival in vitro (Ishii et al., 1993, Neurosci. Lett. 163:159-162), maymediate, in part, induction of neuronal differentiation in MSCs althoughthis surprising result was unexpected based on prior studies.

Nestin, an intermediate filament protein, is expressed inneuroepithelial neuronal precursor stem cells, with expressiondecreasing as the neuron matures. Experimental data shows that when theMSC-differentiated neuronal cells are stained to detect nestin, theexpression of nestin decreases over time (FIGS. 9A-9C). Further,staining for trkA, a high-affinity nerve growth factor receptor which ispresent in neurons, demonstrates that trkA levels remain unchangedthroughout the maturation process of the MSC-differentiated neuronalcell (FIGS. 9D-9F).

To begin examining the hypothesis that the anti-oxidant properties ofBME mediated induction of neuronal differentiation in MSCs, rMSCs weretreated with other anti-oxidants, e.g., dimethylsulfoxide (DMSO),butylated hydroxyanisole (BHA), or butylated hydroxytoluene (BHT),ascorbic acid, dimethylfumarate, n-acetylcysteine, and the like, bothalone and in combination with each other. Further, treatment with theanti-oxidant dithiothreitol (DTT) in combination with BHA also inducedneuronal differentiation by MSCs suggesting that DTT alone may alsoelicit neuronal differentiation.

Each anti-oxidant treatment (e.g., DMSO, BHA, BHT, ascorbic acid,dimethylfumarate, n-acetylcysteine, and the like, both alone and incombination) elicited neuronal morphologies with a time course similarto the effects of BME. Additionally, preliminary data suggested thattreatment using about 2% (v/v) DMSO and about 200 millimolar BHA(DMSO/BHA) was preferred although a wide range of concentrationselicited neuronal differentiation.

To further characterize neuronal identity, MSCs treated with DMSO/BHAwere stained for neurofilament-M (NF-M), a neuron-specific intermediatefilament that helps initiate neurite elongation (Carden et al., 1987,Neurosci. 7:3489-3504). The data disclosed previously elsewhere hereindemonstrated that BME treatment of MSCs caused increased expression ofNF-M in cells exhibiting neuronal morphologies. Most cells displayingrounded cell bodies with processes (arrow) after DMSO/BHA exposureexpressed high levels of NF-M, while flat undifferentiated cells (>) didnot (FIG. 5A). Pre-adsorption of NF-M antibody with purified NF-Mprotein abolished staining (FIG. 5B), establishing specificity.

DMSO/BHA treated cultures were then examined for the presence of tau, aneuron-specific microtubule-associated protein expressed bydifferentiating neurons (Kosik and Finch, 1987, J. Neurosci.7:3142-3153). Cells exhibiting a neuronal morphology (arrow) expressedtau protein in the cell body as well as in the processes (*), whileundifferentiated flat cells were tau-negative (<) (FIGS. 5C and 5D). Thedata disclosed herein indicate that the method described herein induceneuronal differentiation of marrow stromal cells.

Activity-Dependent Synaptic Vesicle Recycling

To further characterize neuronal properties, cultures were treated withthe styryl dye FM1-43, which labels the outer leaflet of synapticvesicles upon activity-dependent transmitter release (Betz and Bewick,1992, Science 255:200-203; Betz et al., 1992, J. Neurosci. 12:363-375;Diefenbach et al., 1999, J. Neurosci. 19:9436-9444). Exposure todepolarizing concentrations of K⁺ resulted in fluorescent labeling ofgrowth cones (

), suggesting that the cells were recycling synaptic vesicles consequentto activity-dependent transmitter release.

Clonal Analysis

To determine whether individual rMSCs exhibit stem cell characteristicsof self-renewal and pluripotentiality, individual clones were analyzed.To establish clones, rMSCs were plated at approximately 10 cells persquare centimeters, grown to 50-150 cells per colony, isolated withcloning cylinders, transferred to separate wells and eventually toindividual flasks. Single cells replicated as typical rMSCs anddifferentiated into NSE-positive neurons after BME treatment.

Analysis of four distinct clonal lines is shown in FIG. 7A-7D. Eachindividual clone generated refractile, process-bearing, NSE-positivecells following BME treatment. Undifferentiated rMSCs (>) andtransitional cells (

) were evident in each clonal line. Therefore, clones derived from asingle cell can give rise to both rMSCs and neurons, indicating stemcell characteristics.

Human Stromal Cells Differentiate into Neurons

The neuronal potential of MSCs was not unique to rodents as demonstratedby the following experiments using MSCs obtained from humans (hMSCs).hMSCs were isolated from a healthy adult donor and grown in vitro(Bjornson et al., 1999, Science 283:534-537). hMSCs resembled theirrodent counterparts, growing as large flat cells in the undifferentiatedstate.

Cells from passage two were subjected to the neuronal differentiationprotocol and stained for NSE or NF-M expression. After BME treatment,hMSCs attained neuronal characteristics and increased NSE expression ina time frame similar to that observed for rMSCs. Contracted cell bodieselaborated processes and stained strongly for NSE expression within 3hours (FIGS. 8A and 8B). Transitional cells were also evident (

). Many processes elaborated by hMSC-derived neurons exhibited terminalbulbs (arrow in 8B), which may represent growth cones. Growth conemorphologies with filopodial extensions (

) were clearly evident on the processes elaborated by paired neuronsdepicted in the image in FIG. 8C. These cells also expressed NF-M,consistent with their neuronal differentiation (FIG. 8D).

The data disclosed herein demonstrate that rat and human MSCs retain thecapacity to differentiate into non-mesenchymal derivatives, specificallyneurons, suggesting that intrinsic genomic mechanisms of commitment,lineage restriction and cell fate are mutable. Environmental signalsapparently can elicit the expression of pluripotentiality that extendswell beyond the accepted fate restrictions of cells originating inclassical embryonic germ layers. These adult cells are bothself-renewing and multipotential (Owens and Friedenstein, 1988, CibaFoundation Symp. 136, Chichester, U.K. pp. 42-60; Prockop, 1997, Science276; 71-74; Ferrari et al., 1998, Science 279:1528-1530; Caplan, 1991,J. Orthop. Res. 9:641-650; Pereira et al., 1995, Proc. Natl. Acad. Sci.USA 92:4857-4861; Kuznetsov et al., 1997, Brit. J. Haemotology97:561-570; Majumdar et al., 1998, J. Cell. Physiol. 176:57-66;Pittenger et al., 1999, Science 284:143-147), thereby fulfilling many ofthe criteria of a stem cell population.

To the best of Applicants' knowledge, this is the first report thatperipheral mesenchymal cells can differentiate into neurons in vitro.Further, the present invention provides, for the first time, methods ofdirecting differentiation of MSCs into neuronal cells in vitro. MSCs areuseful in the treatment of a wide variety of neurologic diseasesdisorders and conditions, and these cells offer significant advantagesover other so-called “stem” cells. That is, bone marrow cells arereadily accessible, obviating the risks of obtaining neural stem cellsfrom the brain, and provide a renewable population which can be expandedin vitro thereby allowing complex gene manipulations to be performed forex vivo gene therapy and/or for cell therapy for CNS diseases, disordersor conditions that require administering cells to a CNS site.Furthermore, autologous transplantation overcomes the ethical andimmunologic concerns associated with the use of fetal tissue. Moreover,MSCs grow rapidly in culture, precluding the need for immortalization,and differentiate into neurons exclusively using the protocols disclosedherein.

Expression of Neuronal Proteins in MSC-Differentiated Neuronal Cells

The data disclosed herein demonstrates that neuronal cellsdifferentiated from MSCs as described herein express variousneuron-related proteins. For example, immunocytochemical analysis ofthese differentiated neurons revealed the expression of beta-3 tubulin.Further, TOAD-64, a neuronal protein of unknown function, is alsodetectable using immunocytochemical techniques, as well assynaptophysin, which is associated with synapses and synaptic vesicles.Using polyclonal and monoclonal antibody-based procedures, these cellshave been demonstrated to express choline acetyltransferase, an enzymeresponsible for the synthesis of the neurotransmitter acetylcholine.Finally, tyrosine hydroxylase, the rate-limiting enzyme in catecholaminebiosynthesis, was also detected immunocytochemically in a population ofthese differentiated neurons.

It is apparent that due to the presence of these neuronal gene products,the differentiated neurons may be therapeutically beneficial to treatingthose diseases affecting cholinergic and catecholaminergic systems, suchas, for example, Alzheimer's disease, Parkinson's disease, orschizophrenia.

Transplantation of the Differentiated Neurons to Experimental Animals

The differentiated neurons generated as described herein, were furthertested to determine their viability in vivo. The neurons weretransplanted, using sterile technique and known and acceptedneurosurgical procedures (1997, Grill et al.; 1995, Gage et al., 1994,Dunnett et al.), into the hippocampus or striatum of the brain or thedorsal horn of the spinal cord of individual rats. Each rat received atransplant to one of the aforementioned areas. The rats were returned totheir cages and received standard postoperative care with access to foodand water ad libatum.

To determine whether neuron viability was maintained in vivo, apost-operative study of the rats receiving the transplant was conducted.Rats receiving the neuronal transplant were examined 42 days after thetransplantation operation took place. Using fluorescence microscopy todetect bisbenzimide-positive transplanted cells, histologic studies ofthe hippocampal and striatal regions of the brain revealed that thetransplanted neurons survived in the hippocampus. This result indicatesthat long-term survival of the transplanted, differentiated neurons ispossible. An examination of the rats receiving the transplanted neuronsin the dorsal horn of the spinal cord demonstrated a survival period ofat least three days. Further, the processes of the transplanted neuronsin this area grew to at least two to three times in length than the cellbody diameter.

As is evident from these results, transplanted, differentiated neuronsexpress many neuronal proteins, retain viability in vivo, and seeminglyexert no detectable deleterious effect on the living animal. As aresult, these neurons create a potential therapeutic treatment for avariety of brain and spinal cord diseases, including, but not limitedto, Alzheimer's disease, Parkinson's disease, Schizophrenia, and spinalcord injury resulting from trauma or degeneration.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A method of inducing differentiation of an isolated marrow stromalcell into a neuronal cell, said method comprising contacting saidisolated marrow stromal cell with at least one neuronaldifferentiation-inducing compound, thereby inducing differentiation ofsaid isolated marrow stromal cell into a neuronal cell.
 2. The method ofclaim 1, wherein said isolated marrow stromal cell is a human cell. 3.The method of claim 1, wherein said neuronal differentiation-inducingcompound is a trophic factor.
 4. The method of claim 1, wherein saidneuronal differentiation-inducing compound is a growth factor.
 5. Themethod of claim 4, wherein said growth factor is selected from the groupconsisting of platelet-derived growth factor, fibroblast growth factor2, and nerve growth factor.
 6. The method of claim 1, wherein saidneuronal differentiation-inducing compound is an anti-oxidant.
 7. Themethod of claim 6, wherein said anti-oxidant is selected from the groupconsisting of beta-mercaptoethanol, dimethylsulfoxide, butylatedhydroxytoluene, butylated hydroxyanisole, ascorbic acid,dimethylfumarate, and n-acetylcysteine.
 8. The method of claim 7,wherein said anti-oxidant is beta-mercaptoethanol.
 9. The method ofclaim 7, wherein said anti-oxidant is dimethylsulfoxide.
 10. The methodof claim 7, wherein said anti-oxidant is dimethylsulfoxide and butylatedhydroxyanisole.
 11. A method of producing an isolated neuronal cell,said method comprising isolating a marrow stromal cell, contacting saidmarrow stromal cell with a neuronal differentiation-inducing compoundwherein said compound induces said isolated marrow stromal cell todifferentiate into an isolated neuronal cell, thereby producing saidisolated neuronal cell.
 12. A method of treating a human patient havinga disease, disorder or condition of the central nervous system, saidmethod comprising obtaining a bone marrow sample from a human donor,isolating stromal cells from said bone marrow sample, inducing saidstromal cells to differentiate into isolated neuronal cells, andadministering said isolated neuronal cells to the central nervous systemof said human patient, wherein the presence of said isolated neuronalcells in said central nervous system of said human patient effectstreatment of said disease, disorder or condition.
 13. The method ofclaim 12, wherein said disease, disorder or condition of the centralnervous system is selected from the group consisting of Alzheimer'sdisease, Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, a tumor, a trauma, elderly dementia, Tay-Sach's disease,Sandhoff's disease, Hurler's syndrome, Krabbe's disease, birth-inducedtraumatic central nervous system injury, epilepsy, multiple sclerosis,trauma, tumor, stroke, and spinal cord injury.
 14. The method of claim12, wherein prior to administering said isolated neuronal cells, saidisolated neuronal cells are transfected with an isolated nucleic acidencoding a therapeutic protein, wherein when said protein is expressedin said cells said protein serves to effect treatment of said disease,disorder or condition.
 15. The method of claim 14, wherein said isolatednucleic acid encodes a therapeutic protein selected from the groupconsisting of a cytokine, a chemokine, a neurotrophin, another trophicprotein, a growth factor, an antibody, and a glioma toxic protein.
 16. Amethod of treating a human patient in need of neuronal cells, saidmethod comprising obtaining marrow stromal cells from a human patient,propagating said marrow stromal cells in culture under conditions thatinduce their differentiation into neuronal cells, transplanting saidneuronal cells into said human patient in need of said neuronal cells,thereby treating said human patient in need of neuronal cells.
 17. Anisolated neuronal cell made by a method of inducing differentiation ofan isolated marrow stromal cell, said method comprising contacting saidisolated marrow stromal cell with at least one neuronaldifferentiation-inducing compound, thereby inducing differentiation ofsaid isolated marrow stromal cell into said neuronal cell.
 18. The cellof claim 17, wherein said cell is a human cell.
 19. An isolated neuronalcell made by a method of inducing differentiation of an isolated marrowstromal cell, said method comprising contacting said isolated marrowstromal cell with at least one neuronal differentiation-inducingcompound, thereby inducing differentiation of said isolated marrowstromal cell in said neuronal cell, wherein said neuronal cell isfurther transfected with an isolated nucleic acid encoding a therapeuticprotein, and further wherein when said protein is expressed in said cellsaid protein serves to effect treatment of a disease, disorder, orcondition of the central nervous system.
 20. The cell of claim 19,wherein said isolated nucleic acid encodes a protein selected from thegroup consisting of a cytokine, a chemokine, a neurotrophin, anothertrophic protein, a growth factor, an antibody, and a glioma toxicprotein.
 21. The cell of claim 19, wherein said cell is a human cell.22. An isolated neuronal cell made by a method of producing an isolatedneuronal cell, said method comprising isolating a marrow stromal cell,contacting said marrow stromal cell with a neuronal differentiationinducing compound, wherein said compound induces said isolated marrowstromal cell to differentiate into said isolated neuronal cell, therebyproducing said isolated neuronal cell.
 23. The cell of claim 22, whereinsaid cell is a human cell.
 24. An isolated neuronal cell made by amethod of producing an isolated neuronal cell, said method comprisingisolating a marrow stromal cell, contacting said marrow stromal cellwith a neuronal differentiation inducing compound, wherein said compoundinduces said isolated marrow stromal cell to differentiate into saidisolated neuronal cell, thereby producing said isolated neuronal cell,wherein said neuronal cell is further transfected with an isolatednucleic acid encoding a therapeutic protein, and further wherein whensaid protein is expressed in said cell, said protein serves to effecttreatment of a disease, disorder, or condition of the central nervoussystem.
 25. The cell of claim 24, wherein said isolated nucleic acidencodes a protein selected from the group consisting of a cytokine, achemokine, a neurotrophin, another trophic protein, a growth factor, anantibody, and a glioma toxic protein.
 26. The cell of claim 25, whereinsaid cell is a human cell.